Isolation and identification of T cells

ABSTRACT

The present invention relates to improved autologous T cell vaccines and improved methods for their production. The invention is also directed to methods for treating autoimmune diseases such as multiple sclerosis or rheumatoid arthritis using autologous T cell vaccines. The invention is further directed to the diagnosis of T cell associated diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationNumber PCT/US2003/024548, filed Aug. 6, 2003, which claims the benefitof U.S. Provisional Application No. 60/402,521, filed Aug. 8, 2002.

FIELD OF THE INVENTION

The present invention relates generally to the field of diagnosis andtreatment of autoimmune disease, such as multiple sclerosis (MS). Moreparticularly, it concerns the isolation of antigen-specific T cells. Inaddition, the present invention concerns the use of antigen-specific Tcells for the treatment of autoimmune disease, such as MS.

BACKGROUND

Intercellular recognition complexes formed by T cell receptors (TCR) oncytotoxic T lymphocytes or T helper cells and MHC/peptide complexes onantigen presenting cells (APC) are a common recognition component in adiverse set of cell-cell encounters that activate T cells both duringthe development of the repertoire of T cells within an individualorganism (positive selection; negative selection; peripheral survival)and during the control (T helper) and effector stages (T killer) of anadaptive immune response.

In the adaptive immune response, antigens are recognized byhypervariable molecules, such as antibodies or TCRs, which are expressedwith sufficiently diverse structures to be able to recognize anyantigen. Antibodies can bind to any part of the surface of an antigen.TCRs, however, are restricted to binding to short peptides bound toclass I or class II molecules of the major histocompatibility complex(MHC) on the surface of APCs. TCR recognition of a peptide/MHC complextriggers activation (clonal expansion) of the T cell.

TCRs are heterodimers composed of two chains which can be αβ(alpha-beta) or γδ (gamma-delta). The structure of TCRs is very similarto that of immunoglobulins (Ig). Each chain has two extracellulardomains, which are immunoglobulin folds. The amino-terminal domain ishighly variable and called the variable (V) domain. The domain closestto the membrane is the constant (C) domain. These two domains areanalogous to those of immunoglobulins, and resemble Fab fragments. The Vdomain of each chain has three complementarity determining regions(CDR). Proximal to the membrane, each TCR chain has a short connectingsequence with a cysteine residue that forms a disulfide bond betweenboth chains.

Genes encoding αβ and γδ heterodimers are only expressed in the T-celllineage. The four TCR loci (α, β, γ and δ) have a germ-line organizationvery similar to that of Ig. α and γ chains are produced byrearrangements of V and J segments whereas β and δ chains are producedby rearrangements of V, D, and J segments. The gene segments for TCRchains are located on different chromosomes, except the δ-chain genesegments that are between the V and J gene segments of the α chain. Thelocation of δ-chain gene segments has a significance: a productiverearrangement of α-chain gene segments deletes C genes of the δ-chain,so that in a given cell the αβ heterodimer cannot be co-expressed withthe γδ receptor.

In mice, there are about 100 Vα and 50 Jα genes segments and only one Cαsegment. The δ-chain gene family has about 10 V, 2 D, and 2 J genesegments. The β-chain gene family has 20-30 V segments and two identicalrepeats containing 1 Dβ, 6 Jβ and 1 Cβ. Finally, the γ-chain gene familycontains 7 V and 3 different J-C repeats. In humans the organization issimilar to that of mice, but the number of segments varies.

The rearrangements of gene segments in α and β chains is similar to thatof Igs. The α chain, like the light chain of Ig is encoded by V, J, andC gene segments. The β chain, like the heavy chain of Ig, is encoded byV, D, and J gene segments. Rearrangements of α chain gene segmentsresult in VJ joining and rearrangements of β chain result in VDJjoining. After transcription of rearranged genes, RNA processing, andtranslation, the α and β chains are expressed linked by a disulfide bondin the membrane of T cells.

TCR gene segments are flanked by recognition signal sequences (RSS)containing a heptamer and a nonamer with an intervening sequence ofeither 12 nucleotides (one turn) or 23 nucleotides (two turn). As inIgs, enzymes encoded by recombination-activating genes (RAG-1 and RAG-2)are responsible for the recombination processes. RAG1/2 recognize theRSS and join V-J and V-D-J segments in the same manner as in Igrearrangements. Briefly, these enzymes cut one DNA strand between thegene segment and the RSS and catalyze the formation of a hairpin in thecoding sequence. The signal sequence is subsequently excised.

The combinatorial joining of V and I segments in α chains and V, D and Jsegments in β chains produces a large number of possible molecules,thereby creating a diversity of TCRs. Diversity is also achieved in TCRsby alternative joining of gene segments. In contrast to Ig, β and δ genesegments can be joined in alternative ways. RSS flanking gene segmentsin β and δ gene segments can generate VJ and VDJ in the β chain, and VJ,VDJ, and VDDJ on the δ chain. As in the case of Ig, diversity is alsoproduced by variability in the joining of gene segments.

Hypervariable loops of the TCR known as complementarity determiningregions (CDRs) recognize the composite surface made from a MHC moleculeand a bound peptide. The CDR2 loops of α and β contact only the MHCmolecule on the surface of APC, while the CDR1 and CDR3 loops contactboth the peptide and MHC molecule. Compared with Ig, TCRs have morelimited diversity in the CDR1 and CDR2. However, diversity of the CDR3loops in TCRs is higher than that of Ig, because TCRs can join more thanone D segment leading to augmented junctional diversity.

The pathogenesis of a number of autoimmune diseases is believed to becaused by autoimmune T cell responses to self-antigens present in theorganism. Not all autoreactive T cells are deleted in the thymus, incontradiction with the clonal selection paradigm. Those T cells withTCRs for a broad spectrum of self-antigens represent part of the normalT-cell repertoire and naturally circulate in the periphery. It isunclear why autoreactive T cells are allowed, during their evolution, toundergo differentiation in the thymus and are accommodated in theperiphery. While their physiological role is not understood, theseautoreactive T cells, when activated, present a potential risk in theinduction of autoimmune pathologies. Autoreactive T cells can also beisolated from normal individuals without the consequences of autoimmunediseases. It has been established that antigen recognition ofautoreactivity by itself is not sufficient to mediate theautodestructive process. One of the prerequisites for autoreactive Tcells to be pathogenic is that they must be activated.

Autoreactive T cells are implicated in the pathogenesis of autoimmunediseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA).The pathogenesis of autoreactive T cells in MS is generally held toarise from T cell responses to myelin antigens, in particular myelinbasic protein (MBP). MBP-reactive T cells are found to undergo in vivoactivation, and occur at a higher precursor frequency in blood andcerebrospinal fluid in patients with MS as opposed to controlindividuals. These MBP-reactive T cells produce T_(H)1 cytokines, e.g.IL-2, TNFα, and γ-interferon (IFNγ), which facilitate migration ofinflammatory cells into the central nervous system and exacerbatemyelin-destructive inflammatory responses in MS.

Myelin-reactive T cells have also been shown to be involved in thepathogenesis of experimental autoimmune encephalomyelitis (EAE) inanimals, which resembles MS. EAE can be induced actively in susceptibleanimals by injecting myelin proteins emulsified in an adjuvant orpassively by injecting myelin-reactive T-cell lines and clones derivedfrom myelin-sensitized animals. When activated in vitro, very smallnumbers of myelin-reactive T cells are required to induce EAE, while100-fold more resting T cells with the same reactivity are incapable ofmediating EAE.

EAE has been shown to be prevented and also treated by vaccination withinactivated myelin-reactive T cells, a procedure called T-cellvaccination (Ben-Nun et al., Nature, 1981; 292: 60-61). T-cellvaccination induces regulatory immune responses comprised ofanti-idiotypic T cells and anti-ergotypic T cells, which lead to thedepletion of myelin-reactive T cells. By depleting myelin-reactive Tcells, therapeutic effects are observed in EAE and other experimentalautoimmune disease models (Lider et al., Science, 1988; 239:820-822;Lohse et al., Science, 1989; 244: 820-822).

Due to the success in autoimmune disease models, T cell vaccination hasrecently advanced to clinical trials in patients with MS. Based on theresults in experimental models such as EAE, it is believed thatdepletion of autoreactive T cells may improve the clinical course of MSand other autoimmune diseases.

In a pilot clinical trial, vaccination with irradiated autologousMBP-reactive T cell clones elicited CD8⁺ cytolytic T cell responses thatspecifically recognized and lysed circulating MBP-reactive T cells(Zhang et al., Science, 1993; 261: 1451-1454, Medaer et al., Lancet1995: 346:807-808). Three subcutaneous inoculations with irradiatedMBP-reactive T cell clones resulted in the depletion of circulatingMBP-reactive T cells in patients with MS.

In a preliminary clinical trial, circulating MBP-reactive T cells weredepleted in relapsing remitting MS patients and secondary progressive MSpatients (Zhang et al., J Neurol., 2002; 249:212-8), by vaccinating thepatients with three subcutaneous injections of irradiated autologousMBP-reactive T cells. T cell vaccination was beneficial to each group ofpatients as measured by rate of relapse, expanded disability scale scoreand MRI lesion activity. However, there was a trend for an acceleratedprogression after about twelve months following the last injection. Thesignificance of the apparent accelerated progression is unknown, but maybe associated with a gradual decline of the immunity induced by T cellvaccination against MBP-reactive T cells. In approximately 10-12% of theimmunized patients, MBP-reactive T cells reappeared at about the sametime as the accelerated progression. In some cases, the reappearingMBP-reactive T cells originated from different clonal populations thatwere not detected before vaccination, suggesting that MBP-reactive Tcells may undergo clonal shift or epitope spreading. Clonal shift ofMBP-reactive T cells has been observed in previous studies (Zhang et al.1995) and may be associated with the on-going disease process.

Although T cell vaccination has been demonstrated to be effective fordepleting myelin-reactive T cells and potentially beneficial for MSpatients, there are several problems with the treatment. T cell vaccinetreatment for each patient must be individualized because the T cellreceptors of myelin-reactive T cells are highly diverse and vary betweendifferent MS patients (Vandevyver et al., Eur. J. Immunol., 1995;25:958-968, Wucherpfennig et al., J. Immunol., 1994; 152:5581-5592, Honget al., J. Immunol., 1999; 163:3530-3538).

In addition to being individualized for each patient, up to 8 weeks isrequired to produce a given T cell vaccine using current procedures.Production of a T cell vaccine begins with isolating mononuclear cellsfrom the cerebrospinal fluid (CSFMCs) or peripheral blood (PBMCs) of apatient. The isolated mononuclear cells are then cultured with myelinpeptides for 7-10 days to activate myelin-reactive T cells. Cultures arethen tested for specific proliferation to myelin peptides by measuring[³H]-thymidine incorporation in the presence of myelin peptides over aperiod of 3 days. Cultures testing positive for specific proliferationto myelin peptides are then serially diluted to obtain clonal T celllines or directly expanded. The cells are then cultured up to 6-8 weeksto expand the T cells. When the final T cell vaccine product is clonal,the T cells are homogenous with a single pattern of Vβ-Dβ-Jβ gene usage.Usually, three to six of the clonal cell lines are combined to produce aheterogeneous formulation with multiple patterns of Vβ-Dβ-Jβ gene usage.When the final T cell vaccine product is produced by direct expansion,the T cells are heterogeneous with more than one pattern of Vβ-Dβ-Jβgene usage.

The individualized nature of T cell vaccination and the prolonged cellculture needed for production of each vaccine make treatment expensiveand labor intensive under current methodologies. The extended timerequired for cell culture also creates a significant risk ofcontamination. Finally, the likelihood of clonal shift or epitopespreading of myelin-reactive T cells may require the subsequentproduction of a T cell vaccine for each patient with a different patternof Vβ-Dβ-Jβ gene usage.

Therefore, there exists a need to develop improved methods of isolatingT cells with specificity for antigens, such as MBP, that may be used toproduce T cell vaccines for the treatment of patients with Tcell-mediated diseases such as MS. There also exists a need to developimproved methods for producing T cell vaccines with a heterogeneouspattern of Vβ-Dβ-Jβ gene usage to account for clonal shift ofautoreactive T cells.

SUMMARY OF THE INVENTION

The present invention is generally directed to methods of isolatingantigen-specific T cells and more particularly T cells specific for selfor autoantigens. The methods for isolating one or more T cells specificfor an antigen of interest generally comprise incubating a samplecomprising T cells obtained from a patient with said antigen or aderivative thereof; selecting one or more T cells that express one ormore first markers selected from the group consisting of CD69, CD4,CD25, CD36 and HLADR; and one or more second markers selected from thegroup consisting of IL-2, IFNg, TNFα, IL5, IL-10 and IL-13.

The methods of the invention are particularly useful for isolatingautoreactive T cells which play a role in the pathogenesis of autoimmunediseases.

The methods of the invention also permit the diagnosis of autoimmunedisease as well as monitoring the progression of the disease and formonitoring the efficacy of treatments for the disease.

The methods of the invention also allow the preparation of autologous Tcell vaccines for the treatment of T cell related autoimmune diseases.

The methods for vaccine preparation generally involve the isolation ofantigen-specific T cells as described above optionally followed bysubsequent culturing steps which allows the expansion of the populationof isolated antigen-specific T cells.

The invention inter alia is also directed to T cell vaccines andpharmaceutical compositions comprising antigen-specific T cells isolatedusing the methods of the invention.

The methods of the invention are also useful for characterizing T cellreceptors and their encoding nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates FACS identification of cells expressing CD69 andγIFN (top) and CD69 and TNFα (bottom) in a multiple sclerosis patientbefore stimulation (left), after stimulation with residues 83-99 of MBP(middle), and after stimulation with residues 83-99 of MBP (right).

FIG. 2 demonstrates FACS identification of cells expressing CD69 andγIFN (top) and CD69 and TNFα (bottom) in a healthy control patientbefore stimulation (left), after stimulation with residues 83-99 of MBP(middle), and after stimulation with residues 83-99 of MBP (right).

DETAILED DESCRIPTION

1. Definitions

To aid in the understanding of the present invention, several terms aredefined below.

“Autoantigen” or “self-antigen” as used herein refers to an antigen orepitope which is native to the mammal which is immunogenic in saidmammal disease is conserved in that species of mammal and which may beinvolved in the pathogenesis of autoimmune.

“CD,” “cluster of differentiation” or “common determinant” as usedherein refers to cell surface molecules recognized by antibodies.Expression of some CDs is specific for cells of a particular lineage ormaturational pathway, and the expression of others varies according tothe state of activation, position, or differentiation of the same cells.

“Derived from” or “a derivative thereof,” in the context of nucleotidesequences means that the nucleotide sequence is not limited to thespecific sequence described, but also includes variations in thatsequence, which may include nucleotide additions, deletions,substitutions, or modifications to the extent that the variations to thedescribed sequence retain the ability to hybridize under moderate orhighly stringent conditions to the complement of the described sequence.In the context of peptide or polypeptide sequences, “derived from” or “aderivative thereof” means that the peptide or polypeptide is not limitedto the specific sequence described, but also includes variations in thatsequence, which may include amino acid additions, deletions,substitutions, or modifications to the extent that the variations in thelisted sequence retain the ability to elicit an immune response to thedescribed sequence.

“Immunogenic,” when used to describe a peptide or polypeptide, means thepeptide or polypeptide is able to induce an immune response, either Tcell mediated, antibody, or both.

“Immune-related disease” means a disease in which the immune system isinvolved in the pathogenesis of the disease. A subset of immune-relateddiseases are autoimmune diseases. Autoimmune diseases include, but arenot limited to, rheumatoid arthritis, myasthenia gravis, multiplesclerosis, psoriasis, systemic lupus erythematosus, autoimmunethyroiditis (Hashimoto's thyroiditis), Graves' disease, inflammatorybowel disease, autoimmune uveoretinitis, polymyositis, and certain typesof diabetes. In view of the present disclosure, one skilled in the artcan readily perceive other autoimmune diseases treatable by thecompositions and methods of the present invention.

“PCR” means the polymerase chain reaction, for example, as generallydescribed in U.S. Pat. No. 4,683,202 (issued Jul. 28, 1987 to Mullis),which is incorporated herein by reference. PCR is an amplificationtechnique wherein selected oligonucleotides, or primers, may behybridized to nucleic acid templates in the presence of a polymerizationagent (such as a DNA or RNA polymerase) and nucleotide triphosphates,whereby extension products may be formed from the primers. Theseproducts may then be denatured and used as templates in a cyclingreaction that amplifies the number and amount of existing nucleic acidswhich may facilitate their subsequent detection. A variety of PCRtechniques are known in the art and may be used in connection with thedisclosure herein.

A “Peptide” or “polypeptide” is a linked sequence of amino acids and maybe natural, synthetic, or a modification or combination of natural andsynthetic.

“Primer” means an oligonucleotide, whether natural, synthetic, or amodification thereof, capable of acting as a point of initiation ofnucleotide synthesis sufficiently complementary to a specific nucleotidesequence on a template molecule.

“Probe” means an oligonucleotide, whether natural, synthetic, or amodification thereof, capable of specifically binding to a sufficientlycomplementary nucleotide sequence.

“T cell mediated disease” means a disease arising as a result of T cellsrecognizing self-antigens.

“Treatment” or “treating,” when referring to protection of an animalfrom a disease, means preventing, suppressing, repressing, or completelyeliminating the disease. Preventing the disease involves administering acomposition of the present invention to an animal prior to onset of thedisease. Suppressing the disease involves administering a composition ofthe present invention to an animal after induction of the disease butbefore its clinical appearance. Repressing the disease involvesadministering a composition of the present invention to an animal afterclinical appearance of the disease.

2. Isolation of Antigen-Specific T Cells

a. Isolation of Monoclonal Antigen-Specific T Cells

T cells may be activated and expanded in cell culture by incubation withan antigen target and antigen presenting cells. Once activated, T cellsundergo a complex cascade of cell signaling which leads to thetranscription and expression of many gene products. The inventiondescribed herein takes advantage of gene products specific for activatedT cells for the identification and isolation of T cells with desiredantigen specificity.

In a first aspect, the present invention is directed to a method forisolating a T cell that is specific for an antigen of interest. A samplecomprising T cells is incubated with a particular antigen, which causesthe activation of a T cell specific for the antigen of interest. Thesample may be incubated with the antigen for 1 to 7 days. The sample mayalso be incubated with the antigen for less than 1 day. The sample mayalso be incubated with the antigen for less than 16 hours. The samplemay also be incubated with the antigen for less than 12 hours. Thesample may also be incubated with the antigen for less than 8 hours. Thesample may also be incubated with the antigen for less than 4 hours. Thesample may also be incubated with the antigen for less than 2 hours.

A T cell specific for the antigen of interest may then be isolated byselecting for T cells that express gene products of T cells activated asdescribed above. Subsets of activated T cells may be isolated byselecting for T cells with subset-specific gene products or cell surfacemarkers (e.g., CD4 vs. CD8).

The antigen of interest includes myelin basic protein (MBP), proteolipidprotein (PLP), myelin oligodendrocyte glycoprotein (MOG), or a fragmentthereof. The antigen of interest may also be an immunodominant fragmentincluding, but not limited to, residues 83-99 or residue 151-170 of MBP.The antigen of interest may also be a combination of two or moreindividual antigens of interest.

The antigen or a derivative thereof, used to activate the T cells may beany immunogen which is capable of eliciting an immune response to theantigen of interest. The activating antigen may be the antigen ofinterest, or a derivative thereof. The activating antigen may be MBP,PLP, MOG, or a fragment and/or derivative thereof. The activatingantigen may also be an immunodominant fragment including, but notlimited to, residues 83-99 or residue 151-170 of MBP, or a fragmentand/or derivative thereof. The activating antigen may also be acombination of two or more individual activating antigens. Theactivating antigen may be used one or more times to activate T cellsspecific for an antigen of interest

The T cells may be present in any sample comprising mononuclear cells.The sample may be isolated from the peripheral blood or cerebral spinalfluid of an MS patient or from the synovial fluid of a RA patient. Tcells from patients with other autoimmune diseases may be similarlyisolated from peripheral blood and/or tissues involved with the disease.Mononuclear cells may be enriched in the sample by using centrifugationtechniques known to those in the art including, but not limited to,Ficoll® gradients. T cells may also be enriched in the sample by usingpositive selection, negative selection, or a combination thereof forexpression of gene products of T cells.

The gene product for identifying or negatively selecting for activated Tcells may be a cell surface marker or cytokine, or a combinationthereof. Cell surface markers for identifying activated T cells include,but are not limited to, CD69, CD4, CD8, CD25, HLA-DR, CD28, and CD134.CD69 is an early activation marker found on B and T lymphocytes, NKcells and granulocytes. CD25 is an IL-2 receptor and is a marker foractivated T cells and B cells. CD4 is a TCR coreceptor and is marker forthymoctes, T_(H)1- and T_(H)2-type T cells, monocytes, and macrophages.CD8 is also a TCR coreceptor and is marker for cytotoxic T cells. CD134is expressed only in activated CD4⁺ T cells.

Cell surface markers for negatively selecting for activated T cellsinclude, but are not limited to, CD36, CD40, and CD44. CD28 acts as astimulatory T-cell activation pathway independent of the T-cell receptorpathway and is expressed on CD4⁺ and CD8⁺ cells. CD36 is a membraneglycoprotein and is a marker for platelets, monocytes and endothelialcells. CD40 is a marker for B cells, macrophages and dendritic cells.CD44 is a marker for macrophages and other phagocytic cells. Subsets ofT cells may be isolated by using positive selection, negative selection,or a combination thereof for expression of cell surface gene products ofhelper T cells or cytotoxic T cells (e.g., CD4 vs. CD8).

Cytokines for identifying activated T cells of the present inventioninclude, but are not limited to cytokines produced by T_(H)1-type Tcells (cell-mediated response) and T_(H)2-type T cells (antibodyresponse). Cytokines for identifying activated T_(H)1-type T cellsinclude, but are not limited to, IL-2, gamma interferon (γIFN) andtissue necrosis factor alpha (TNFα). Cytokines for identifying activatedT_(H)2-type T cells include, but not limited to, IL-4, IL-5, IL-10 andIL-13. Subsets of T cells may also be isolated by using positiveselection, negative selection, or a combination thereof for expressionof cytokine gene products of helper T cells or cytotoxic T cells (e.g.,γIFN vs. IL4).

An activated T_(H)1-type T cell specific for an antigen of interest maybe isolated by identifying cells that express CD69, CD4, CD25, IL-2,IFNγ, TNFα, or a combination thereof. An activated T_(H)1-type T cellspecific for an antigen of interest may also be isolated by identifyingcells that express CD69 and CD4 together with IFNγ or TNFα. An activatedT_(H)2-type T cell specific for an antigen of interest may be isolatedby identifying cells that express CD69, CD4, IL-4, IL-5, IL-10, IL-13,or a combination thereof. A combination of an activated T_(H)1-type Tcell and a T_(H)2-type T cell specific for an antigen of interest may beisolated by identifying cells that express CD69, CD4, CD25, IL-2, IFNγ,TNFα, or a combination thereof and cells that express CD69, CD4, IL-4,IL-5, IL-10, IL-13, or a combination thereof/

The gene products used for positive or negative selection of theactivated T cells of the present invention may be identified byimmunoselection techniques known to those in the art which utilizeantibodies including, but not limited to, fluorescence activated cellsorting (FACS), magnetic cell sorting, panning, and chromatography.Immunoselection of two or more markers on activated T cells may beperformed in one or more steps, wherein each step positively ornegatively selects for one or more markers. When immunoselection of twoor more markers is performed in one step using FACS, the two or moredifferent antibodies may be labeled with different fluorophores.

Magnetic cell sorting may be performed using super-paramagneticmicrobeads composed of iron oxide and a polysaccharide coat. Preferablythe microbeads may be approximately 50 nanometers in diameter, and havea volume about one-millionth that of a typical mammalian cell. Themicrobeads are preferably small enough to remain in colloidalsuspension, which permits rapid, efficient binding to cell surfaceantigens. The microbeads preferably do not interfere with flowcytometry, are biodegradable, and have negligible effects on cellularfunctions. The antibody coupling to the microbeads may be direct orindirect, via a second antibody to a ligand such as fluorescein.

The antibody may be of classes IgG, IgM, IgA, IgD, and IgE, or fragmentsor derivatives thereof, including Fab, F(ab′)₂, Fd, and single chainantibodies, diabodies, bispecific antibodies, bifunctional antibodiesand derivatives thereof. The antibody may be a monoclonal antibody,polyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to an epitope of a geneproduct specific for activated T cells, or a sequence derived therefrom.The antibody may also be a chimeric antibody.

The antibody may be derivatized by the attachment of one or morechemical, peptide, or polypeptide moieties known in the art that allowthe identification and/or selection of the activated T cell to which theantibody is bound. The antibody may be conjugated with a chemical moietysuch as a fluorescent dye. An activated T cell bound by a fluorescentlylabeled antibody may be isolated using techniques including, but notlimited to, fluorescence activated cell sorting (FACS). The antibody mayalso be conjugated with a magnetic particle, such as a paramagneticmicrobead (Miltenyi Biotec, Germany). An activated T cell bound by amagnetically labeled antibody may be isolated using techniquesincluding, but not limited to, magnetic cell sorting.

For cell-surface expressed gene products, the antibody may directly bindto the gene product and may be used for cell selection. For cell-surfacegene products expressed at low concentrations, magnetofluorescentliposomes (Scheffold, et al. Nature Med 6:107-110, 2000) may be used forcell selection. At low levels of expression, conventional fluorescentlylabeled antibodies may not be sensitive enough to detect the presence ofthe cell surface expressed gene product. Fluorophore-containingliposomes may be conjugated to antibodies with the specificity ofinterest, thereby allowing detection of the cell surface expressed geneproduct.

For intracellular gene products, such as cytokines, the antibody may beused after permeabilizing the cells. Alternatively, to avoid killing thecells by permeabilization, the intracellular gene product if it isultimately secreted from the cell may be detected as it is secretedthrough the cell membrane using a “catch” antibody on the cell surface.The catch antibody may be a double antibody that is specific for twodifferent antigens: (i) the secreted gene product of interest and (ii) acell surface protein. The cell surface protein may be any surface markerpresent on T cells, in particular, or lymphocytes, in general, (e.g.,CD45). The catch antibody may first bind to the cell surface protein andthen bind to the intracellular gene product of interest as it issecreted through the membrane, thereby retaining the gene product on thecell surface. A labeled antibody specific for the captured gene productmay then be used to bind to the captured gene product, which allows theselection of the activated T cell (Manz, et al. Proc. Natl. Acad. Sci.USA 92:1921-1925, 1995, incorporated herein by reference).

Certain forms of cytokines are also found expressed at low concentrationon the cell surface. For example, γIFN is displayed at a lowconcentration on the cell surface with kinetics similar to those ofintracellular γIFN expression (Assenmacher, et al. Eur J. Immunol, 1996,26:263-267). For forms of cytokines expressed on the cell surface,conventional fluorescently labeled antibodies or fluorophore containingliposomes may be used for detecting the cytokine of interest. One ofordinary skill in the art will recognize other techniques for detectingand selecting extracellular and intracellular gene products specific foractivated T cells.

The T cells isolated by the present invention may be enriched by atleast 90% from whole blood. The T cells may also be enriched by at least95% from whole blood. The T cells may also be enriched by at least 98%from whole blood. The T cells may also be isolated at least 99.5% fromwhole blood.

b. Isolated Monoclonal Antigen-Specific T Cells

In a second aspect, the present invention is directed to a T cellspecific for an antigen of interest isolated by the method of the firstaspect of the present invention.

c. Isolation of Polyclonal Antigen-Specific T Cells

In a third aspect, the present invention is directed to a method forisolating T cells that are specific for one or more antigens ofinterest. A sample comprising T cells is incubated with one or moreantigens, which cause the activation of T cells specific for one or moreantigens. T cells specific for one or more antigens may then be isolatedas in the first aspect of the present invention.

The T cells may have a heterogeneous pattern of Vβ-Dβ-Jβ gene usage thatexpress different TCRs which are each specific for an antigen ofinterest. The T cells may also have a heterogeneous pattern of Vβ-Dβ-Jβgene usage that express different TCRs which are specific for more thanone antigen of interest. As described below, T cells comprising aheterogeneous pattern of Vβ-Dβ-Jβ gene usage may be used to formulate apolyclonal T cell vaccine which may prevent epitope spreading invaccinated patients.

d. Isolated Polyclonal Antigen-Specific T Cells

In a fourth aspect, the present invention is directed to T cellsspecific for one or more antigens of interest isolated by the method ofthe third aspect of the present invention.

3. Quantifying the Number of Antigen-Specific T Cells

In a fifth aspect, the present invention is directed to a method ofdetermining the relative frequency of T cells specific for one or moreantigens of interest in a sample by determining the number of T cellsisolated by the method of the first or third aspects of the presentinvention.

4. Diagnosing an Autoimmune Disease

In a sixth aspect of the present invention, a patient with an autoimmunedisease may be diagnosed by obtaining a sample from a patient andisolating autoreactive T cells by the method of the first or thirdaspects of the present invention. The autoimmune disease may bediagnosed by comparing the level of autoreactive T cells in a patient toa control. The level of autoreactive T cells may be determined inaccordance with the method of the fifth aspect of the present invention.

5. Monitoring the Progress of an Autoimmune Disease

In a seventh aspect of the present invention, an autoimmune disease maybe monitored by determining the frequency of autoreactive T cells in asample from a patient with an autoimmune disease in accordance with thefifth aspect of the present invention. The severity of symptoms of theautoimmune disease may correlate with the number of autoreactive Tcells. In addition, an increase in the number of autoreactive T cells inthe sample may be used as an indication to apply treatments intended tominimize the severity of the symptoms and/or treat the disease beforethe symptoms appear.

6. Producing a Vaccine for the Treatment of an Autoimmune Disease

In an eighth aspect of the present invention, a composition may beproduced for treating an autoimmune disease by inactivating autoreactiveT cells which have been isolated (and optionally expanded in culture asdescribed herein) by the method of the first or third aspects of thepresent invention. The autoreactive T cells may be inactivated using anumber of techniques known to those in the art including, but notlimited to, chemical inactivation or irradiation. The autoreactive Tcells may be preserved either before or after inactivation using anumber of techniques known to those in the art including, but notlimited to, cryopreservation. As described below, the composition may beused as a vaccine to deplete autoreactive T cells in autoimmunepatients.

The composition may be a pharmaceutical composition, which may beproduced using methods well known in the art. Pharmaceuticalcompositions used as preclinical and clinical therapeutics in thetreatment of disease or disorders may be produced by those of skill,employing accepted principles of diagnosis and treatment. Suchprinciples are known in the art, and are set forth, for example, inBraunwald et al., eds., Harrison's Principles of Internal Medicine, 11thEd., McGraw-Hill, publisher, New York, N.Y. (1987), which isincorporated by reference herein. The pharmaceutical composition may beadministered to any animal which may experience the beneficial effectsof the composition. Animals receiving the pharmaceutical composition maybe humans or other mammals.

a. Vaccine

In a ninth aspect, the present invention is drawn to a compositionproduced by the method of the eighth aspect of the present invention.The composition may be a vaccine, which may be used to depleteautoreactive T cells in autoimmune patients.

7. Treatment of an Autoimmune Disease

In a tenth aspect, an autoimmune disease may be treated in patients withautoreactive T cells by administering a composition according to theninth aspect of the present invention. The composition may be a vaccine,which may lead to the depletion of autoreactive T cells in the patient.

A vaccine may comprise autoreactive T cells comprising homogeneous(“monoclonal”) or heterogeneous (“polyclonal”) patterns of Vβ-Dβ-Jβ geneusage. Clinical studies indicate that autoimmune patients receivingautologous monoclonal T cell vaccination may show a gradual decline inthe immunity against myelin-reactive T cells. In some cases, thereappearing autoreactive T cells may originate from different clonalpopulations, suggesting that myelin-reactive T cells may undergo clonalshift or epitope spreading potentially associated with the ongoingdisease process. Clonal shift or epitope spreading may be a problem inautoimmune diseases mediated by autoreactive T cells. A vaccinecomprising polyclonal autoreactive T cells capable of depleting multiplepopulations of autoreactive T cells may avoid problems with clonal shiftor epitope spreading.

The composition may be a pharmaceutical composition, which isadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intraarterial, intradermal, intramuscular, intraperitoneal, transdermal,transmucosal, intracerebral, intrathecal, or intraventricular routes.Alternatively, or concurrently, administration may be by the oral route.The pharmaceutical compositions may be administered parenterally bybolus injection or by gradual perfusion over time.

The dosage administered may be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. The dose ranges forthe administration of the pharmaceutical compositions may be largeenough to produce the desired effect, whereby, for example, autoreactiveT cells are depleted, as measured by the seventh aspect of the presentinvention, is achieved, and the autoimmune disease is significantlyprevented, suppressed, or treated. The doses may not be so large as tocause adverse side effects, such as unwanted cross reactions,generalized immunosuppression, anaphylactic reactions and the like.

The pharmaceutical compositions may further comprise suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which may facilitate processing of the active compositionsinto preparations which can be used pharmaceutically. Additives to thepharmaceutical compositions may include the inclusion of an adjuvant,such as alum, chitosan, or other adjuvants known in the art. (See, forexample, Warren et al., Ann. Rev. Immunol. 4:369-388 (1986); Chedid, L.,Feder. Proc. 45:2531-2560 (1986), which are incorporated herein byreference). The pharmaceutical compositions may also further compriseliposomes to enhance delivery or bioactivity, using methods andcompounds known in the art.

Suitable formulations for parenteral administration include aqueoussolutions of the inactivated autoreactive T cells, for example,water-soluble salts in aqueous solution. In addition, oil suspensionscomprising inactivated autoreactive T cells may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Thesuspension may also contain stabilizers.

The inactivated autoreactive T cells may be formulated usingconventional pharmaceutically acceptable parenteral vehicles foradministration by injection. These vehicles may be nontoxic andtherapeutic, and a number of formulations are set forth in Remington'sPharmaceutical Sciences, (supra). Nonlimiting examples of excipients arewater, saline, Ringer's solution, dextrose solution and Hank's balancedsalt solution. Pharmaceutical compositions may also contain minoramounts of additives such as substances that maintain isotonicity,physiological pH, and stability.

The inactivated autoreactive T cells may be formulated at total cellconcentrations including from about 5×10² cells/ml to about 1×10⁹cells/ml. Preferred doses of the inactivated autoreactive T cells foruse in preventing, suppressing, or treating an autoimmune disease may bein the range of about 2×10⁶ cells to about 9×10⁷ cells.

8. Determination of TCR Repertoire

In an eleventh aspect, the present invention is drawn to a method ofdetermining the repertoire of nucleic acids encoding one or more T cellreceptors, or a portion thereof, in an autoimmune patient by amplifyingnucleic acids encoding one or more T cell receptors from T cellsisolated by the first or third aspects of the present invention, whereinsaid amplification is performed using a primer pair. The first primer ofthe primer pair may be an oligonucleotide of about 15 to 30 nucleotidesin length that hybridizes to a nucleic acid comprising the variableregion of the TCR gene. The second primer of the primer pair may be anoligonucleotide of about 15 to 30 nucleotides in length that hybridizesto a nucleic acid comprising the constant region of the TCR gene. Theprimer pair may be used to amplify a nucleic acid that hybridizes to theVβ-Dβ-Jβ region of the TCR gene.

Nucleic acids encoding one or more T cell receptors from T cells (the“Target Sequence”) or a fragment thereof may be amplified from a sampleby the polymerase chain reaction (PCR) using any particular PCRtechnique or equipment known in the art. For example, PCR amplificationmay follow a procedure wherein a reaction mixture is prepared thatcontains the following ingredients: 5 μL 10×PCR buffer II (100 mMTris-HCl, pH 8.3, 500 mM KCl), 3 μL 25 mM MgCl₂, 1 μL 10 mM dNTP mix,0.3 μL Taq polymerase (5 U/μL) (AmpliTaq Gold, Perkin Elmer, Norwalk,Conn.), 30 pmol of a first primer, 30 pmol of a second primer, and 1 μLof sample DNA. The polymerase may be stable at temperatures of at least95° C., have a processivity of 50-60 and have an extension rate ofgreater than 50 nucleotides per minute.

The PCR reaction may be performed with an amplification profile of 1 minat 95° C. (denaturation), 20 sec at 56° C. (annealing), and 40 sec at72° C. (extension) for a total of 40 cycles. Before the first cyclebegins, the reaction mixture may undergo an initial denaturation for aperiod of about 5 min to 15 min. Similarly, after the final cycle iscomplete, the reaction mixture may undergo a final extension for aperiod of about 5 min to 10 min. Certain PCR reactions may work with asfew as 15 to 20 cycles or as many as 50 cycles. Depending upon thespecific PCR reaction, longer or shorter incubation times and higher orlower temperatures for each step of the amplification profile may beused.

The sample comprising the Target Sequence, may be a nucleic acid, suchas genomic DNA, cDNA, DNA previously amplified by PCR, or any other formof DNA. The sample may be isolated, directly or indirectly, from anyanimal or human tissue comprising T cells, such as peripheral bloodmononuclear cells (PBMC). Genomic DNA may be isolated directly from atissue comprising T cells. cDNA may be isolated indirectly by reversetranscription of mRNA directly isolated from a tissue comprising Tcells.

The ability to detect the Target Sequence may be enhanced by isolatingthe sample DNA indirectly by amplification of genomic DNA, cDNA, or anyother form of DNA, by a two-step PCR reaction. For example, a first PCRamplification reaction may be performed to amplify a preliminaryfragment that is larger than, and comprises, a fragment to which thefirst and second primers are capable of selectively binding on oppositestrands. A second PCR amplification reaction may then be performed,using the preliminary fragment as a template with the first and secondprimers, to amplify a fragment comprising the Target Sequence. If eitherthe first or second primer is used in the first PCR reaction to amplifythe preliminary fragment, the second PCR reaction is “semi-nested.” Ifneither the first or second primer is used in the first PCR reaction toamplify the preliminary fragment, the second PCR reaction is “nested.”

In an exemplary two-step PCR reaction, one or more nucleic acidsencoding one or more T cell receptors from T cells may be amplified byperforming a first PCR reaction using a first preliminary primer thatanneals to the Vβ region of the TCR gene and a second preliminary primerthat anneals to the Cβ region of the TCR gene, which amplifies apreliminary fragment that extends from Vβ through the Vβ-Dβ-Jβ junctionto Cβ, followed by a second PCR reaction which may be nested orsemi-nested. In light of the present disclosure, the skilled artisanwill be able to select appropriate primers and reaction conditions forPCR amplification of the Target Sequence.

After amplification of the Target Sequence, the amplified product may bedetected by a number of procedures. For example, an aliquot ofamplification product may be loaded onto an electrophoresis gel, towhich an electric field is applied to separate DNA molecules by size. Inanother method, an aliquot of amplification product may be loaded onto agel stained with SYBR green, ethidium bromide, or another molecule thatwill bind to DNA and emit a detectable signal. A dried gel may contain alabeled oligonucleotide that hybridizes to the Target Sequence, fromwhich an autoradiograph may be taken by exposing the gel to film.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Isolation of Myelin-Reactive T Cells for T Cell Vaccination

1. Preparation of PBMC and the Primary Stimulation

Fresh blood specimens from MS patients and control patients wereprocessed within 2 hours of collection. Alternatively, mononuclear cellsmay be obtained from the cerebrospinal fluid (CSFMCs) of MS patients.Peripheral blood mononuclear cells (PBMCs) were isolated from the wholeblood by standard Ficoll gradient separation method. Specifically,heparinized blood was diluted with Hanks balanced salt solution (HBSS)(1:1 blood/HBSS) and then slowly laid over the Ficoll-hypaque solutionin a centrifuge tube and centrifuged for 20 minutes at 1800 rpm, 18° C.to 25° C., with no brake. PBMCs were then washed by adding excess HBSSand centrifuged at 1700 rpm for 10 minutes at 18° C. to 25° C. PurifiedPBMCs were washed three times in RPMI 1640 medium by centrifugation andsubsequently resuspended in AIM V medium (Gibco, Grand Island, N.Y.).Cell number was counted and cells were plated onto 96-well U-bottomedculture plates at a density of 200,000 cells/well. All plates werelabeled with patient number and patient initials. The cells wereincubated at 37° C. in the presence of synthetic peptides listed inTable 1 corresponding to the known immunodominant regions of threemyelin proteins, myelin basic protein (P), proteolipid protein (PLP),and myelin oligodendrocyte glycoprotein (MOG) at a concentration of 20μg/ml. Plates were placed in a CO₂ incubator at 37° C. and visuallyinspected daily. Cells were cultured for 7-10 days without change ofculture medium to selectively grow antigen-specific T cells.

TABLE 1 Activating Peptides Myelin Antigen Peptides Amino Acid SequencesMyelin basic ENPVVHFFKNIVTPRTP SEQ ID NO.1 protein, peptide-1 (MBP-1)Myelin basic SKIFKLGGRDSRSGSPMARR SEQ ID NO.2 protein, peptide-2 (MBP-2)Proteolipid protein, LFCGCGHEALTGTEKLIETY SEQ ID NO.3 peptide-3 (PLP-3)Proteolipid protein, WTTCQSIAFPSKTSASIGSL SEQ ID NO.4 peptide-4 (PLP-4)Myelin GQFRVIGPRHPIRALVG SEQ ID NO.5 oligodendrocyte glycoprotein,peptide-6 (MOG-6) Myelin EVELPCRISPGKNATGMEVGW SEQ ID NO.6oligodendrocyte glycoprotein, peptide-7 (MOG-7)2. Identification and Selection of Antigen-Specific T Cells

The cells described above are then selected for the expression of geneproducts indicative of activated T cells. See Section 2(a) above. ACytokine Catch Reagent (Miltenyi Biotec) (as described above) is used inorder to detect the intracellular cytokine γIFN or TNFα when ultimatelyexcreted from the cell. Briefly, the Cytokine Catch Reagent (typically abispecific antibody which binds to both the activated T cell marker andthe secreted cytokine) is incubated first with the cells at 4-8° C. inorder to bind to the CD45 molecule on the cell surfaces or otheractivated T cell surface marker such as CD69. The cells with the boundCytokine Catch Reagent are then incubated at 37° C. for 45 minutes toallow the γIFN or TNFα within the cell to also bind to the CytokineCatch Reagent as the cytokine is secreted from inside the cell duringthis incubation period. γIFN or TNFα, now bound to the cell surface bythe Cytokine Catch Reagent which is then detected using an antibodyspecific for cytokine of interest conjugated to the fluorochrome PE.

The cell surface molecules CD4 and CD69, are detected using antibodiesconjugated to different fluorochromes. The CD4⁺ cell population isselected first by gating and then, within this population, the“double-positive” (CD69 and IFNγ or CD69 and TNFα) stained cells areseparated by FACS and collected aseptically.

The isolated myelin-reactive T cells are then directly expanded bystimulating with rIL-2, PHA, anti-CD3 or other general T cell mitogen inthe presence of irradiated autologous PBMCs for 7-10 days.Myelin-reactive T cells lines are propagated in culture until the totalcell number reached approximately 20 million.

EXAMPLE 2 Diagnosis of MS

Two to 100 ml of blood are collected from the patient and one or moresynthetic peptides are added directly to the whole blood to prime, orstimulate, the T lymphocytes. The peptides correspond to the knownimmunodominant regions of three myelin proteins, myelin basic protein(MBP), proteolipid protein (PLP), and myelin oligodendrocyteglycoprotein (MOG). The blood is incubated with the peptides for 1 to 7days to activate the myelin-specific T cells. At the end of thisantigen-priming period, the cells are re-challenged with antigens in ashort re-stimulation assay.

Myelin peptide-activated T cells are detected by permeabilizing the cellmembrane with a detergent solution, washing the cells, then incubatingwith one or more staining antibodies to detect CD4 or CD69 molecules onthe cell surface or IFNγ or TNFα intracellularly. The stainingantibodies are conjugated to different fluorochromes so that theyfluoresce at different wavelengths when excited by a 488 nanometer laserby FASC analysis. The population of CD4⁺ T cells is selected first byusing the CD4⁺ cells for gating and then within this population, thecells that are immunoreactive with both antibodies (CD69 and γIFN orCD69 and TNFα) are identified. This population of “double-positive”myelin-reactive T cells has been shown to increase significantly inmultiple sclerosis (MS) patients as compared to healthy controls in asimilar study (FIG. 1, MS patient, FIG. 2, healthy control). Using thismethod, the number of myelin-reactive T cells circulating in the bloodof a patient may be determined before, during and after treatment todetermine the effect of an MS therapy on the autoreactive T cellpopulation. The endpoint may be determined as either the percentage ofdouble-positive stained cells or as the absolute number ofdouble-positive stained cells.

EXAMPLE 3 Diagnosis of MS Using Antibody-Conjugated Liposomes

Whole blood is obtained from a patient and stimulated with one or moresynthetic peptides as described in Example 2. The blood is incubatedwith the peptides for 3 to 16 hours to activate the myelin-specific Tcells. At the end of this antigen-priming period, the cells may bere-challenged with antigens in a short re-stimulation assay prior tostaining with magnetofluorescent liposomes conjugated to antibodiesagainst IFNγ, TNFα, or a combination thereof. The cells are also stainedwith an antibody to CD4 and/or CD69. The stained myelin-reactive T cellsare detected as described in Example 2.

EXAMPLE 4 Determination of TCR Clonal Repertoire

The T cell receptor (TCR) clonal repertoire represented in a cellpopulation may be analyzed by isolating out the double-positive stainedcell population by cell sorting as described in Example 2 and Example 3.DNA is extracted from the isolated cells and used to performquantitative polymerase chain reaction (PCR) assays usingoligonucleotide primers specific for 25 known TCR variable beta chain(Vβ) gene families. This procedure yields information on thedistribution of TCR Vβ gene usage and indicates the clonality ofpathogenic T cell populations. This method may also be used to determineif clonal or epitopic shifting of the myelin-reactive T cell populationis occurring in an MS patient.

EXAMPLE 5 The Depletion of Myelin-reactive T Cells by T Cell Vaccination

Patients with relapsing-remitting (RR)-MS and secondary-progressive(SP)-MS received three subcutaneous injections of irradiated autologousmyelin-reactive T cell clones isolated by direct expansion, with threeadditional injections 4, 12 and 20 weeks thereafter. Patients weremonitored for changes in the precursor frequency of myelin-reactive Tcells, rate of relapse, expanded disability status score (EDSS) and MRIlesion activities over a period of 24 months. The results were comparedwith pre-vaccination values in a self-paired manner. In addition, theclinical data of the placebo arms of RR-MS in the beta-interferon-1aclinical trial (Jacobs et al., 1996) and SP-MS in a recent beta-IFN-1bstudy (European Study Group, Lancet, 352:1491-1497 (1998)) were includedto provide natural history data of MS for comparison. The T cellfrequency was either undetectable or substantially declined aftervaccination at week 20. The results confirmed depletion ofmyelin-reactive T cells by T cell vaccination in patients with MS.

EXAMPLE 6 Vaccination of MS Patient Using Autologous Myelin-Reactive TCells

The vaccination protocol is similar to that used in previous clinicalstudies (Zhang et al., 1993, Medaer et al., 1995). Briefly,myelin-reactive T cell clones prepared according to Example 1 areactivated with phytohemagglutinin (PHA) (4 μg/ml) in the presence ofirradiated PBMCs as a source of accessory cells. Cells are then culturedfor 10 days in RPMI 1640 media supplemented with 10% heat-inactivatedhuman AB serum and 100 units per mL of rIL-2. Activated myelin-reactiveT cells are subsequently washed three times with sterile saline toremove residual PHA, rIL-2 and cell debris and finally resuspended in 2ml of saline. After irradiation (10,000 rads, ¹³⁷Ce source), the cellsare injected subcutaneously on two arms (1 ml/arm). The number of Tcells used for vaccination range from 40×10⁶ to 80×10⁶ cells perinjection and are chosen by an extrapolation of T cell doses effectivein experimental animals on the basis of relative skin surface areas(Ben-Nun et al., 1981). Each patient receives two subcutaneousinjections followed by repeat injections at 4, 12 and 20 weeks.

Patients are then observed for time to onset of confirmed progression ofdisability, EDSS, rate of relapse and MRI lesion activities. The resultsare compared with the patient's own pre-treatment course as well as theplacebo arms of two recent clinical trials in RR-MS and SP-MS patients,which serve as an estimate of the natural history of MS (Jacobs et al,1996), European Study Group, 1998). Time to progression is determined byan increase of at least 1.0 on the EDSS (Poser et al., 1983) persistingfor at least 2 months. On-study exacerbations are defined by theappearance of new neurological symptoms or worsening of preexistingneurological symptoms lasting for at least 48 hours, accompanied byobjective change on neurological examination (worsening of at least 0.5point on EDSS). Patients are instructed to report events between thescheduled regular visits, and are examined by a neurologist if symptomssuggested an exacerbation. Safety assessments included adverse events,vital signs and physical examinations at regular visits. The differencesin the clinical variables in study patients before and after T cellvaccination are analyzed using the Wilcoxon's rank-sum test.

EXAMPLE 7 Alteration of Clinical Course of MS After Vaccination

Patients receive T-cell vaccinations prepared according to Example 1with no adverse effects. The mean EDSS declines in patients with RR-MSover a period of 24 months after vaccination. By comparison, there is anincrease of mean EDSS by 0.61 in the natural history of RR-MS (n=56)over the same period of observation, as was reported in a trialconducted using beta-IFN-1a trial (Jacobs et al., 1996). In addition,the proportion of the patients that have either unchanged or improvedEDSS is higher than that of the natural MS history. Few, if any,patients in the treated RR-MS group progress beyond EDSS of 2.0 within24 months as compared to 18% of patients in the natural history of MS.

In the SP-MS cohort, mean EDSS progresses slower over a period of 24months as compared to +0.6 recorded in the natural history of SP-MS(European Study Group, Lancet 1998; 352:1491-1497). Furthermore,estimation of time to confirmed progression using the Kaplan-Meiermethod shows considerable delay as compared to the natural history of MSpatients (20% progression in 12 months for RR-MS and 9 months for SP-MS)(Jacobs et al., Ann. Neurol, 1996; 39:285-294, European Study Group,1998).

1. An autologous T-cell vaccine consisting of inactivated T cells thatare reactive against SEQ ID NOS: 1-6.
 2. An autologous T cell vaccinecomprising inactivated T cells elicited from a patient sample comprisingmononuclear cells by a combination of antigens consisting of SEQ IDNOS:1-6.
 3. The T cell vaccine according to claim 1, wherein the T cellsare inactivated by irradiation.
 4. The T cell vaccine according to claim2, wherein the T cells are inactivated by irradiation.
 5. The T cellvaccine according to claim 2, wherein the patient sample comprisesperipheral blood T cells.
 6. The T cell vaccine according to claim 2wherein the patient sample comprises cerebrospinal fluid T cells.
 7. Amethod of treating multiple sclerosis comprising administering to apatient in need thereof the autologous T cell vaccine according toclaim
 1. 8. A method of treating multiple sclerosis comprisingadministering to a patient in need thereof the autologous T cell vaccineaccording to claim 2.